ImProm-II™ Reverse Transcription System (Cat.# A3800) combines a newly formulated reverse transcriptase, an optimized reaction buffer and the associated reagents qualified for efficient synthesis of first-strand cDNA. The system is designed to be used for quantitative synthesis of high-quality cDNA in preparation for gene-specific analysis such as by PCR. The optimized ImProm-II™ Reaction Buffer enables robust activity by both ImProm-II™ Reverse Transcriptase and Promega Taq DNA Polymerase. The buffer also combats the inhibiting effect of reverse transcriptases on thermophilic DNA polymerases, which is often encountered in RT-PCR (1)
The ImProm-II™ Reverse Transcription System can be used for coupled RT-PCR or uncoupled RT-PCR; therefore, the system is suitable for the approach dictated by your experimental questions. The components of the ImProm-II™ System are provided separately, giving the greatest flexibility in performing first-strand cDNA synthesis, coupled or uncoupled to PCR amplification. The individual packaging also allows for better optimization in the cDNA synthesis step (2)
In this article the ImProm-II™ System is used in combination with Taq DNA Polymerase for single-tube, coupled RT-PCR. Methods are defined for RNA isolation, first-strand cDNA synthesis and gene-specific amplification and analysis.
As evident in Figure 1, fewer than five copies of a transcript were detected using the ImProm-II™ Reverse Transcription System supplemented with Taq DNA Polymerase in coupled RT-PCR. Kanamycin Positive Control RNA (1.2kb) was titrated over 11 orders of magnitude (from approximately 1010 to a single copy. We combined aliquots of the diluted RNA template with gene-specific primers for kanamycin-specific RT-PCR. In parallel, we combined aliquots of the diluted RNA template with gene-specific primers plus oligo(dT) to demonstrate that oligo(dT) can be used for cDNA priming with no deleterious effect on the quality or the RT-PCR.
Figure 2 shows that the ImProm-II™ System can be used to detect a medium-abundance message (γ-actin) in 1pg of total RNA when used with Taq DNA Polymerase for coupled RT-PCR. Human Jurkat total RNA, which had been prepared using the SV Total RNA Isolation System (Cat.# Z3100), was titrated over a range of approximately 100ng to 0.1pg.
We demonstrated the extremely sensitive detection and proportional response using the ImProm-II™ Reverse Transcription System for coupled RT-PCR. The single-species, polyadenylated kanamycin transcript could be amplified from fewer than five copies of starting material, and medium-abundance γ-actin message could be detected from as little as 1pg of human Jurkat total RNA. This optimized application, a modification of the uncoupled RT-PCR protocol provided in the ImProm-II™ System Technical Manual (#TM236), offers a simple method for sensitive analysis of RNA sequences.
The ImProm-II™ Reverse Transcription System (Cat.# A3800) and the supplied protocol (Technical Manual #TM236) were used with the modifications detailed in this article.
1. Template Preparation: The SV Total RNA Isolation System (Cat.# Z3100) was used to isolate total RNA from human Jurkat cells according to the system instructions (#TM048). Kanamycin RNA (1.2kb) was used as a single-species, poly(A)+ RNA transcript for template titrations of a single target. For all RNA titrations, RNA was serially diluted into ice-cold, Nuclease-Free Water. For each 20µl of RT-PCR, we combined aliquots containing known amounts of diluted RNA with primers in thin-walled, nuclease-free tubes on ice and then added 5µl of RNA plus primer mix to each reaction. RNA-primer combinations as outlined in Table 1 were used to illustrate the success of RT-PCR using gene-specific primers alone or in combination with oligo(dT). The no-template, negative control reactions consisted of water and primer but no RNA. Finally, we heated the RNA/primer mixture at 70°C for 5 minutes and immediately chilled the tubes on ice.
Table 1 lists the RNA targets, oligonucleotide primers and expected product sizes for these experiments.
Table 1. Target RNA and Gene-Specific Oligonucleotide Primers Used.
Notes: Kanamycin Positive Control RNA: 0.1µg, 2.5fmol (~1010 copies) to 2.5ymol (~1 copy). Kanamycin Control Primers: 20pmol each. Oligo(dT)15 Primer (Cat.# C1101): 0.5mg. Human γ-Actin mRNA, medium abundance (0.4%) (3)
. Human Jurkat Cell Total RNA: 100ng to 100ag. Human γ-Actin Primers: 20pmol each.
2. Coupled RT-PCR: For each RT-PCR set (20µl per reaction), we prepared an RT-PCR mix (in 1.5ml nuclease-free tube on ice) containing components of the ImProm-II™ Reverse Transcription System plus Promega Taq DNA Polymerase, minus the RNA template + primers. We mixed the combination gently and kept it on ice. Next, we transferred 15µl of RT-PCR Mix to each prepared 5µl target/primer combination (on ice). We topped each reaction volume with nuclease-free mineral oil to prevent evaporation. The setup is listed in Table 2.
Notes: RNA, maximum 1µg per reaction; primers, 0.4–1µM.
We transferred tubes to a programmed, controlled temperature block for coupled reverse transcription and amplification as follows: Annealed primer and template, 25°C for 5 minutes; first-strand extension, 42°C for 60 minutes; heat inactivation, 95°C for 5 minutes; amplification cycle, (see figure legend for each experiment) and final extension, 70°C for 5 minutes. Table 3 enumerates important observations in designing successful coupled RT-PCR using ImProm-II™ Reverse Transcriptase and Taq DNA Polymerase.
Table 3. Observations About Coupled RT-PCR Using the ImProm-II™ System.
Note: For additional information on optimizing first-strand synthesis, see the Technical Manual for the ImProm-II™ Reverse Transcription System (#TM236).